Alginate hydrogels functionalized with ß-cyclodextrin as a local paclitaxel delivery system.

Modification of drug delivery materials with beta-cyclodextrins (ß-CyD) is known to increase solubility of poorly water-soluble drugs, protect drugs from degradation and sustain release. In this study, we developed a hydrogel drug delivery system for local paclitaxel delivery using the natural polysaccharide alginate functionalized with ß-CyD-moieties. Paclitaxel was chosen due to its ability to form inclusion complexes with cyclodextrins. The rheological and mechanical properties of the prepared hydrogels were characterized, as well as in vitro release of the paclitaxel and in vitro activity on PC-3 prostate cancer cells. Introduction of ß-CyD-moieties into the hydrogel reduces the mechanical properties of the gels compared to nonmodified gels. However, gelation kinetics were not markedly different. Furthermore, the ß-CyD-modified alginate helped to reduce undesired crystallization of the paclitaxel in the gel and facilitated paclitaxel diffusion out of the gel network. Remarkably, the ß-CyD grafted alginate showed increased capacity to complex paclitaxel compared to free HPß-CyD. Release of both paclitaxel and degradation products were measured from the gels and were shown to have cytotoxic effects on the PC-3 cells. The results indicate that functionalized alginate with ß-CyDs has potential as a material for drug delivery systems.

Alginates induce differentiation and expression of CXCR7 and CXCL12/SDF-1 in human keratinocytes--the role of calcium.

Alginates from seaweed are used in chronic wound management, though the molecular and cellular effects of various alginate dressings are not well documented. We have developed ultrapure sodium-alginates from Pseudomonas fluorescens with different content and distribution of single guluronic acid (G) residues (0-45% G), and tested their biological activities on human primary keratinocytes (KCs). The alginates inhibited KC migration and induced expression of differentiation markers. The potency of the alginates correlated with the increasing percentage of single G residues. These findings were explained by different binding and release of ionic calcium (Ca++) from the alginates which subsequently triggered differentiation. Ca-free alginates had no effect on KC migration and differentiation, but the chemokine receptor CXCR7 was upregulated. Q-PCR revealed that also CXCL12/SDF-1, one of two known CXCR7-ligands, was induced by the alginates. Both CXCR7 and CXCL12-induction was dependent on the alginate G-content, and highest upregulation was induced by an alginate with 19% single G residues. In the epidermis, CXCR7 expression was restricted to the basal layer. This study defines two biological effects of ultrapure alginates on KCs, both being dependent on the alginate structure, and being either dependent or independent of Ca.

Characterization of the hydrolysis mechanism of polyalternating alginate in weak acid and assignment of the resulting MG-oligosaccharides by NMR spectroscopy and ESI-mass spectrometry.

Alginate with long strictly alternating sequences of mannuronic (M) and guluronic (G) acid residues, F(G) = 0.47 and F(GG) = 0.0, was prepared by incubating mannuronan with the recombinant C-5 epimerase AlgE4. By partial acid hydrolysis of this PolyMG alginate at pH values from 2.8 to 4.5 at 95 degrees C, alpha-L-GulpA-(1-->4)-beta-D-ManpA (G-M) linkages were hydrolyzed far faster than beta-D-ManpA-(1-->4)-alpha-L-GulpA (M-G) linkages in the polymer chain. The ratio of the rates (kG-M/kM-G) decreased with increasing pH. The dominant mechanism for hydrolysis of (1-->4)-linked PolyMG in weak acid was thus proved to be an intramolecular catalysis of glycosidic cleavage of the linkages at C-4 by the undissociated carboxyl groups at C-5 in the respective units. The higher degradation rate of G-M than M-G glycosidic linkages in the polymer chain of MG-alginate at pH 3.5 and 95 degrees C was exploited to make oligomers mainly consisting of M on the nonreducing and G on the reducing end and, thus, a majority of oligomers with an even number of residues. The ratio of the rate constants kG-M/kM-G at this pH was 10.7. The MG-hydrolysate was separated by size exclusion chromatography and the MG oligosaccharide fractions analyzed by electrospray ionization-mass spectrometry together with 1H and 13C NMR spectroscopy. Chemical shifts of MG-oligomers (DP2-DP5) were elucidated by 2D 1H and 13C NMR.

Construction and analyses of hybrid Azotobacter vinelandii mannuronan C-5 epimerases with new epimerization pattern characteristics.

The secreted mannuronan C-5 epimerases from Azotobacter vinelandii form a family of seven homologous modular type enzymes, which appear to have evolved through duplications and point mutations in the individual modules. The catalytic A modules of these enzymes are responsible for generating the characteristic sequence distribution patterns of G residues in the industrially important polymer alginate by epimerizing M (beta-D-mannuronic acid) moieties to G (alpha-L-guluronic acid). Forty-six different hybrid enzymes were constructed by exchanging parts of the sequences encoding the A modules of AlgE2 (generates consecutive stretches of G residues) and AlgE4 (generates alternating structures). These hybrid enzymes introduce a variety of new monomer-sequence patterns into their substrates, and some regions important for the subsite specificity or processivity of the enzymes were identified. By using time-resolved NMR spectroscopy, it became clear that the rates for introducing alternating structures and consecutive stretches of G residues are different for each enzyme, and that it is the ratio between these rates that determines the overall epimerization pattern. These findings open up new possibilities in biotechnology and in studies of the many biological functions of alginates.